High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR

Goodman, Laura B.; Anderson, Renee R.; Slater, Marcia; Ortenberg, Elen; Renshaw, Randall W.; Chilson, Brittany D.; Laverack, Melissa A.; Beeby, John S.; Dubovi, Edward J.; Glaser, Amy L.

1. Clean the outside of each sample container with 10% bleach and dry thoroughly. Wipe gloved finger tips with 10% bleach between samples to prevent cross-contamination. 2. Place nasal or deep pharyngeal swabs in any sterile, sealed vial (such as a red top blood collection tube) with several drops of saline added to prevent desiccation prior to processing. NOTE: Cotton, plastic, wood-handled, and Dacron and other synthetic swabs are all acceptable, but avoid calcium alginate swabs. 3. For swabs and tracheal washes, add Dulbecco's Modified Eagle Medium (DMEM) so that there is at least 1-2 ml of liquid. Vortex the swab and DMEM vigorously in the tube. Then, use a pipette to transfer approximately 1 ml of media to a new tube. 4. Mechanically lyse tissues (100-200 mg in 1 ml of DMEM) using a tissue disruptor according to the manufacturer's instructions followed by centrifugation for 3 min at 825 x g. Transfer 500 µl of the supernatant to a new tube. 5. For feces, combine 400 mg of feces with 800 µl of 1x phosphate-buffered saline pH 7.4 (PBS). Vortex the suspension for 1 min or longer until the sample is homogenized or completely suspended. Once homogenized, centrifuge the suspension for 10 min at 18,000 x g, then transfer 400 µl to a new tube. 6. For whole uncoagulated blood, vortex the sample and make a 250 µl aliquot. Add six drops of lytic reagent and vortex to mix. Incubate 15 min at 37 °C.